Normal retinal function requires constant replacement of the membranous disks which comprise the outer segments of the photoreceptor cells. During this process disk membranes at the apical ends of the segments detach in packets which are ultimately phagocytosed by the retinal pigment epithelium (RPE). Dysfunction in outer segment phagocytosis by the RPE leads to retinal degeneration and blindness in experimental animals and may be an important contributor to retinal degenerative diseases in humans. The overall objective of this proposal is to characterize the molecular mechanisms by which retinal pigment epithelium recognize and phagocytose effete photoreceptor outer segments. Major emphasis will be on CD36, an 88,000MW glycoprotein that we have shown in preliminary studies to be expressed on RPE in a developmentally regulated manner, and to function as a receptor for rod outer segments (ROS). Specific aims are to use immunohistochemical, immunocytochemical, western blot, and rt-PCR to demonstrate that human RPE express CD36 protein and mRNA. Monoclonal anti- CD36 IgG, soluble cD36, and CD36 cDNA transfected cells will be used to demonstrate that CD36 is a receptor for ROS on RPE. Recombinant CD36/GST fusion proteins will be used to identify the ligand binding domain on CD36 for ROS. The ligand for CD36 on ROS will also be identified, as will other receptors that might be associated with CD36 in this function; e.g. alpha(vn)beta(3) integrin, type I/II scavenger receptors, or advanced glycation end product (AGE)-receptors. The mechanism of CD36-mediated ROS internalization will also be explored. Approaches will include electron microscopy to define the ultrastructural pathway of CD36-mediated internalization. Immunological and mutagenesis approaches will be used to define the role of caveolae and CD36-caveolin interactions in internalization. Similarly the role of CD36 interactions with cytoskeletal elements and non-receptor protein tyrosine kinases in internalization will be studied. The RCS rat is a well described animal model of retinal degeneration caused by defective RPE phagocytosis of ROS. Studies will be done to determine if the ROS uptake and internalization defect in the RCS rat is the result of an abnormality in the CD36 pathway. CD36 expression and molecular form in the RCS rat RPE will be studied as will CD36- mediated signalling pathways. Understanding the molecular details of CD36 function on RPE will provide insight into normal retinal processes such as RPE polarity and RPE phagocytosis, as well as into retinal degenerative states. Novel strategies to diagnose and treat retinal degeneration may thus result from this work.